Sanitary article comprising a microbe-inhibiting composition

ABSTRACT

A sanitary article ( 1 ) such as a sanitary napkin, a panty liner, an incontinence protector, a diaper, an incontinence pad, a feminine insert, a tampon, or the like includes a top sheet ( 2 ) and a microbe-inhibiting composition ( 8 ). The microbe-inhibiting composition ( 8 ) includes at least one extracellular product of at least one probiotic bacterium and/or at least one probiotic bacterium and at least one additive in the form of an organic acid, with at least one of its pKa value not exceeding 5.5, and/or a salt thereof.

TECHNICAL FIELD

The present invention relates to a sanitary article, such as sanitarynapkins, panty liners, incontinence protectors, diapers, tampons, or thelike, that comprises an extracellular product of at least one probioticbacterium and/or at least one probiotic bacterium and at least one ofthe selected additive giving a synergistic inhibitory effect on Candidaalbicans or other unwanted microorganisms.

BACKGROUND OF THE INVENTION

The urogenital area harbors a complex microbial ecosystem comprisingmore than 50 different bacterial species (Hill et al., Scand. J. Urol.Nephrol. 1984; 86 (suppl.) 23-29). The dominating species for fertilewomen in this area are lactic acid producing bacteria belonging to thegenus Lactobacillus. These lactic acid producing members are importantfor retaining a healthy microbial flora in these areas, and act asprobiotic bacteria with an antagonistic effect against pathogenicmicrobial species. Lactic acid producing bacteria inhibit growth andcolonization by other microorganisms by occupying suitable niches forcolonization, by forming biofilms and competing for available nutrients,thereby excluding colonization by harmful microorganisms. Also, theproduction of hydrogen peroxide, specific inhibiting substances, such asbacteriocines, and organic acids (including lactic acid and acetic acid)that lower the pH, inhibit the growth and colonization by othermicroorganisms.

The microbial ecosystem of a healthy individual can be disturbed by theuse of antibiotics, during hormonal changes, such as during pregnancy oruse of contraceptives with estrogen, during menstruation, aftermenopause, in people suffering from diabetes etc. Also, microorganismsmay spread from the anus to the urogenital area, this result in adisturbance of the normal microbial flora and leaves the individualsusceptible to microbial infections such as vaginitis, candidainfections, urinary tract infections and skin infections. Microorganismscommonly associated with these kinds of infections belong to the generaEscherichia, Enterococcus, Pseudomonas, Proteus, Klebsiella,Streptococcus, Staphylococcus, Gardnerella and Candida. Women are atparticular risk due to their shorter distance between the anus and theurogenital tract; specially at risk are young women, who not yet have awell developed microflora in the urogenital area and older women, who inmost cases no longer have a protective flora.

Similarly to the urogenital area, the skin is colonized by an array oforganisms, which forms its normal flora. The numbers and identity of theorganisms vary between different skin sites. This, together with theskin's structural barrier, provides the host with an excellent defenseagainst invading microbes. The number of bacteria on the skin variesfrom a few hundred per cm² on the arid surfaces of the forearm and back,to tens of thousands per cm² on the moist areas such as the axilla andgroin. This normal flora plays an important role in preventing foreignorganisms from colonizing the skin, but it too needs to be kept incheck, in order to avoid skin infections.

Staphylococcus aureus is the most common cause of minor skin infections,such as boils or abscesses, as well as more serious post-operative woundinfection. Treatment involves drainage and this is usually sufficientfor minor lesions, but antibiotics may be given in addition when theinfection is severe and the patient has fever.

Toxic shock syndrome is a systemic infection caused by S. aureus strainswhich produce toxic shock syndrome toxin. The disease came to prominencethrough its association with tampon use by healthy women, but it is notconfined to women and can occur as a result of S. aureus infection atnon-genital sites.

Other common skin infections are caused by Streptococcus pyogenes (groupA streptococci). The organisms are acquired through contact with otherpeople with infected skin lesions and may first colonize and multiply onnormal skin prior to invasion through minor breaks of the epithelium andthe development of lesions.

Treatment with penicillin or erythromycin may be necessary to combat theinfection.

Candida likes skin sites which are moist and warm and also rapidlycolonizes damaged skin. Hence, the relative dryness of most areas ofskin prevents the growth of Candida, which therefore are found in lownumbers on healthy skin. Candida also colonizes the oral and vaginalmucosa and over-growth may result in disease in these sites. C. albicansis associated with diaper dermatitis. A study has shown that C. albicansinduced lesions are remarkably influenced by pH, a lower skin pH givingless lesions (B. Runeman, Acta Derm Venereol 2000; 80: 421-424).

One way to reduce the problems with the kind of infections describedabove is to have a good personal hygiene. However, excessive use ofcleaning agents not only decreases the amount of harmful microbes, butcan harm the beneficial microbial flora, again render it susceptible forpathogenic species to colonize and cause infections. Alternatively,administration of lactic acid producing bacteria to the urogenital areaand the skin, in order to out-compete pathogenic species and facilitatereestablishment and maintenance of a beneficial microbial flora in theseareas, has been found to be a successful means to treat and preventmicrobial infections

It has been suggested that lactic acid producing bacteria can bedelivered via absorbent products, such as diapers, sanitary napkin,incontinence guards, panty liners and tampons, as described in, forexample, WO 92/13577, WO 97/02846, WO 99/17813, WO 99/45099 and WO00/35502.

Other ways of delivering the lactic acid producing bacteria have alsobeen suggested, such as a hygiene tissue that allows both cleaning andcaring of the skin and urogenital area and delivery of probiotic lacticacid producing bacteria, for example, WO 04/060416.

A major problem with providing products intended to be used for transferof lactic acid producing bacteria, is that the bacteria have to retainviability during transport and storage of the products. Lactic acidproducing bacteria rapidly lose viability under semi moist conditions,and it is therefore important that the bacteria are not exposed tomoisture during storage. One way to partly overcome this problem inabsorbent products provided with lactic acid producing bacteria has beento supply the products with the bacteria, drying said products to removemost of the moisture in them and providing the product in moistureimpervious packages (W099/17813).

WO 00/61201 discloses a sanitary product containing an effective amountof a viable, non-pathogenic, probiotic bacterium, such as Bacilluscoagulans, or an extracellular product thereof.

EP 1140226 describes the combination of a pH regulating substance in theform of a partially neutralized superabsorbent material with lacticacid-producing bacteria.

In view of the prior art there is still a need for sanitary articleswith an improved prebiotic and/or probiotic effect, which articles areeasy to store and transport.

SUMMARY OF THE INVENTION

In view of this prior art it is an object of the present invention toprovide a sanitary article comprising a microbe-inhibiting compositionwith an enhanced probiotic and/or prebiotic effect. It is also an objectof the present invention that said sanitary article comprising amicrobe-inhibiting composition is easily transported and stored withoutthe microbe-inhibiting composition losing its micro-flora balancing andhealth promoting function.

The above defined problems are solved by the present invention by asanitary article such as sanitary napkins, panty liners, incontinenceprotectors, diapers, tampons or the like comprising a microbe-inhibitingcomposition comprising an extracellular product of at least oneprobiotic bacterium and/or at least one probiotic bacterium and at leastone additive, in the form of an organic acid, having a pKa value notexceeding 5.5, and/or a salt thereof, giving a synergistic probioticand/or prebiotic effect.

In one aspect said pKa value does not exceed 5.

According to the present invention said microbe-inhibiting extracellularproduct is a supernatant obtained by filtration or centrifugation of aculture of a probiotic bacterium.

In one embodiment of the invention said microbe-inhibiting compositionis substantially free from probiotic bacteria, preferably not present inan amount higher than 100 CFU/ml and more preferably not higher than 10CFU/ml.

In another embodiment of the invention said microbe-inhibitingcomposition comprises at least one probiotic bacterium and at least oneof said additives.

In a further embodiment of the invention said microbe-inhibitingcomposition comprises at least one probiotic bacterium, an extracellularproduct of at least one probiotic bacterium and at least one of saidadditives.

In one aspect of the invention the probiotic bacterium is a lactic acidproducing bacterium.

In a further aspect of the invention the lactic acid producing bacteriumis Lactobacillus plantarum LB 931. In one other aspect of the inventionthe lactic acid producing bacterium is Lactobacillus fermentum Ess-1. Ina further aspect of the invention the lactic acid producing bacteria isa combination of Lactobacillus plantarum LB 931 and Lactobacillusfermentum Ess-1.

In one aspect of the invention said additive is chosen from acetic acid,propionic acid, lactic acid, ascorbic acid, phenylalanine, citric acid,butyric acid, valeric acid, capronic acid, succinic acid and/or a saltthereof. Preferably said additive is chosen from acetic acid, propionicacid, lactic acid, phenylalanine, citric acid or succinic acid, ascorbicacid, and/or a salt thereof. Most preferably said additive is chosenfrom ascorbic acid, acetic acid, propionic acid, succinic acid and/or asalt thereof.

In another aspect of the invention said salt is a sodium salt;preferably sodium propionate or sodium acetate.

Since the microbe inhibiting composition of the invention combining aprobiotic bacterium and/or an extracellular product thereof and anadditive gives an unexpected synergistic effect in inhibition ofpathogenic microorganisms, a sanitary article according to the inventioncomprising this microbe inhibiting composition has an enhanced probioticand/or prebiotic effect. In addition a sanitary article of the presentinvention is easy to transport and store without the microbe inhibitingcomposition losing its beneficial properties.

DESCRIPTION OF THE DRAWINGS

FIG. 1. shows the growth of C. albicans in extracellular product of LB931 with addition (50 mM) of different acids/salts.

FIG. 2 a. shows the growth of E. coli in extracellular product of LB 931with addition (50 mM) of different acids/salts.

FIG. 2 b. shows the growth of S. Saprofyticus in extracellular productof LB 931 with addition (50 mM) of different acids/salts.

FIG. 3 a. shows the growth of Candida albicans when extracellularproduct of the Lactobacillus fermentum Ess-1 was combined with theadditive ascorbic acid.

FIG. 3 b. shows the growth of Candida albicans when extracellularproduct of the Lactobacillus fermentum Ess-1 was combined with theadditive propionic acid.

FIG. 4 a is a plan view of a sanitary article according to the presentinvention.

FIG. 4 b is a cross sectional view through the sanitary articleaccording to the line II-II in FIG. 4 a.

FIG. 5 a is a plan view of a sanitary article according to the presentinvention.

FIG. 5 b is a cross sectional view through the sanitary articleaccording to the line II-II in FIG. 5 a.

DEFINITIONS

Probiotics/probiotic in the present context relates to livemicroorganisms that confer a health benefit when administered inadequate amounts to a host.

By “lactic acid producing bacteria” is a meant a bacterium producinglactic acid. Preferred lactic acid producing bacteria for the object ofthe present invention include bacteria from the genera Lactobacillus,Lactococcus and Pediococcus. Preferably the selected bacterium used isfrom the species Lactococcus lactis, Lactobacillus fermentum,Lactobacillus rhamnosus, Lactobacillus acidophilus or Lactobacillusplantarum. More preferably the bacterial strain is selected fromLactobacillus plantarum or Lactobacillus fermentum. Even more preferablythe lactic acid producing bacterium is Lactobacillus plantarum LB 931(deposition No. (DSMZ): DSM 11918) and Lactobacillus fermentum Ess-1,(deposition No. (DSMZ): DSM 17851).

Prebiotics/prebiotic in the present contexts relates to substances thatpromote a balanced micro flora when administered in adequate amounts toa host. Examples are nutrients for probiotic bacteria, substances thatpromote adhesion to the host for probiotic bacteria, pH-regulatingsubstances and extracellular products from probiotic bacteria.

By microbe-inhibiting is meant the inhibition of growth, colonizationand/or survival of other microorganisms.

By extracellular product is meant products secreted in cultures ofprobiotic bacteria which extracellular products have an antimicrobialeffect. The extracellular product is obtained from the bacterial cellcultures by e.g. filtration, centrifugation, or both of these, and theresulting supernatant comprising the extracellular product possessesantimicrobial activity useful in a microbe-inhibiting composition.

The term “sanitary article” refers to products that are placed againstthe skin of the wearer to absorb and contain body exudates, like urine,faeces and menstrual fluid, which articles also can be used to deliverprobiotic bacteria to these areas. The invention mainly refers todisposable sanitary articles, which means articles that are not intendedto be laundered or otherwise restored or reused as a sanitary articleafter use. Examples of disposable sanitary articles include femininehygiene products such as sanitary napkins, panty liners, sanitarypanties, feminine inserts and tampons; diapers and pant diapers forinfants and incontinent adults; incontinence pads; diaper inserts andthe like.

DETAILED DESCRIPTION

The present invention pertains to solve the problem of growth,colonization and/or survival of pathogenic microorganisms in theurogenital area and/or in a sanitary article during use thereof.

This problem is in the present invention solved by applying anextracellular product of at least one probiotic bacterium and/or atleast one probiotic bacterium and at least one additive, in the form anorganic acid having a pKa value not exceeding 5.5 and/or a salt thereof,together forming a microbe-inhibiting composition, to a sanitaryarticle.

It should be noted that of course a combination of two or more probioticbacterial strains may be used to produce the extracellular product ofthe invention. Also, it is of course possible to use a combination of atleast two probiotic bacterial strains in a microbe-inhibitingcomposition according to the present invention.

The prior art discloses sanitary articles only comprising probioticbacteria or extracellular products thereof. However themicrobe-inhibiting effect is not always completely satisfying and thereare many problems to overcome when producing and storing sanitaryarticles containing viable micro-organisms as previously discussed.

In the present invention it has been found that by adding an additive,in the form an organic acid having a pKa value not exceeding 5.5,preferable not exceeding 5, and/or a salt thereof, to a probioticbacteria or extracellular products thereof, there is an surprisinglylarge increase in the inhibition of Candida albicans and several othermicro-organisms such as for example E. coli and S. Saprofyticus.

The present invention in some embodiments also aims to decrease theproblems with retaining the microbe-inhibiting effect of the sanitaryarticle during storage. Since all the effect in some embodiments and atleast parts of the effect in other embodiments comes from a compositioncomprising substantially no living organisms, the problem of keeping themicro-organisms viable and thus functional is eliminated or decreased.

FIGS. 4 and 5 show an exemplary embodiment of a sanitary napkin 1 whichcomprises a liquid permeable topsheet 2, a backsheet 3 and an absorbentstructure 4 enclosed there between. The liquid permeable topsheet 2 canbe composed of a nonwoven material, e g spunbonded, meltblown, carded,hydroentangled, wetlaid etc. Suitable nonwoven materials can be composedof natural fibers, such as woodpulp or cotton fibres, manmade fibres,such as polyester, polyethylene, polypropylene, viscose etc. or from amixture of natural and manmade fibres. The topsheet material may furtherbe composed of tow fibres, which may be bonded to each other in abonding pattern, as e.g. disclosed in EP-A-1 035 818. Further examplesof topsheet materials are porous foams, apertured plastic films etc. Thematerials suited as topsheet materials should be soft and non-irritatingto the skin and be readily penetrated by body fluid, such as urine ormenstrual fluid.

The backsheet 3 may consist of a thin plastic film, e.g. a polyethyleneor polypropylene film, a nonwoven material coated with a liquidimpervious material, a hydrophobic nonwoven material, which resistsliquid penetration. Laminates of plastic films and nonwoven materialsmay also be used. The backsheet material is preferably breathable so asto allow vapour to escape from the absorbent structure, while stillpreventing liquids from passing through the backsheet material.

The topsheet 2 and the backsheet 3 have a somewhat greater extension inthe plane than the absorbent structure 4 and extend out side the edgesthereof to form projecting portions 5. The layers 2 and 3 are connectedto each other within the projecting portions 5, e g by gluing or weldingby heat or ultrasonic. The topsheet and/or the backsheet may further beattached to the absorbent structure by any method known in the art, suchas adhesive or welding by heat or ultrasonic. The absorbent structuremay also be unattached to the topsheet and/or the backsheet.

Fastening means in the form of a region 6 of an adhesive is provided onthe side of the backsheet facing away from the wearer during use. Theadhesive may releasably attach to the undergarment of the wearer. Arelease paper 7 protects the adhesive region 6 before use. The adhesiveregion 6 may have any suitable configuration, such as elongate ortransverse strips, dots, full-coated areas etc.

In other embodiments (not illustrated) of sanitary articles according tothe invention other types of fasteners, like friction fasteners, tapetabs or mechanical fasteners like hook-and-loop fasteners etc may beused to fasten the articles to the underwear or around the waist of thewearer. Some sanitary articles are in the form of pants and therefore donot need special fastening means. In other cases the sanitary article isworn in special elastic pants without the need for additional fasteners.

The absorbent structure 4 can be of any conventional kind. Examples ofcommonly occurring absorbent materials are cellulosic fluff pulp, tissuelayers, highly absorbent polymers (so called superabsorbents), absorbentfoam materials, absorbent nonwoven materials or the like. It is commonto combine cellulosic fluff pulp with superabsorbents in an absorbentstructure. It is also common to have absorbent structures comprisinglayers of different material with different properties with respect toliquid acquisition capacity, liquid distribution capacity and storagecapacity. This is well-known to the person skilled in the art and doestherefore not have to be described in detail. The thin absorbent bodies,which are common in today's sanitary articles, often comprise acompressed mixed or layered structure of cellulosic fluff pulp andsuperabsorbent. The size and absorbent capacity of the absorbentstructure may be varied to be suited for different uses such as sanitarynapkins, panty liners, adult incontinence pads and diapers, babydiapers, pant diapers, etc. In some cases the sanitary articles are inthe form of a feminine insert or an incontinence insert withsubstantially no absorbing function at all, the main function for thesearticle is instead to carry and possibly deliver a for example healthpromoting substance. Examples on inserts are a nonwoven layer, aperforated plastic film or a foam layer, or laminates thereof, allpreferably with means for attachment to a garment. Said insert canoptionally comprise a back sheet 3.

An object of the present invention is to provide sanitary articles, suchas sanitary napkins, panty-liners, diapers, incontinence guards etc.suitable for absorbing bodily fluids and simultaneously release amicrobe-inhibiting composition 8 that is to be transferred to the skin,or alternatively, in order to inhibit the growth of unwantedmicroorganisms in the sanitary article itself during use of the article.

It is understood that the sanitary article described above and shown inthe drawings only represents non-limiting examples and that the presentinvention is not limited thereto, but can be used in any type ofsanitary articles as defined above.

The probiotic bacteria which are suitable for use in the presentinvention produce in most cases acid and are in all casesnon-pathogenic. There are many suitable bacteria identified hereinbelow, although the invention is not limited to currently knownbacterial species and strains as long as the above function and theobjectives of the probiotic bacteria are fullfilled.

An important characteristic of the probiotic lactic acid producingbacteria is their capability to produce lactic acid and in some casesalso other acids, said lactic acid increases the acidity of the skinmucosa which helps in preventing the growth, colonization and survivalof undesired fungi and bacteria. Thus, by the mechanism of acidproduction, these probiotic bacteria inhibit the growth of competing andharmful bacteria and fungi. Further important characteristics of saidbacteria is their ability to produce hydrogen peroxide and other microbeinhibiting substances and also their ability to adhere to cell surfacesand thereby prevent adhesion of other harmful bacteria and fungi tothese surfaces.

Typical probiotic bacteria useful in a microbe-inhibiting composition ofthis invention are all members of the Lactobacillus, Lactococcus orPediococcus, which are efficient lactic acid producers, and alsoincluding non-pathogenic members of the Bacillus genus, all members ofthe Bifidobacterium genus, and Pseudomonas limbergii, although certainspecies are especially preferred as described below.

Preferred members of the Lactobacillus genus include Lactobacillusacidophilus, Lactobacillus bulgaricus, Lactobacillus casei,Lactobacillus cereale, Lactobacillus delbrukeii, Lactobacillusfermentum, Lactobacillus gaserii, Lactobacillus jensenii, Lactobacillusplantarum, Lactobacillus rhamnosus, Lactobacillus salivarius,Lactobacillus thermophilus, Lactobacillus paracasai sp. paracasai,Lactobacillus crispatus, Lactobacillus helveticus, Lactobacillus lactis,and the like.

Particularly preferred is the novel bacterium Lactobacillus fermentumEss-1 and Lactobacillus plantarum LB 931. LB 931 has previously beenfound valuable for preventing and/or treating urogenital infections asit inhibits growth of a large number of pathogenic microorganisms, ine.g. EP1060240. Lactobacillus fermentum Ess-1 (deposition No. (DSMZ):DSM 17851) was deposited according to the Budapest Treaty at DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH (Mascheroder Weg 1b,D-38124 Braunschweig) (depositor Essum AB, Box 3160, SE 90304 Ume{dotover (a)}, Sweden, deposited on Jan. 6, 2006).

It should be noted that although in the examples below where only twoLactobacilli strains have been used, these bacterial strains should onlybe seen as a model for probiotic bacteria useful in the practice of thepresent invention. Therefore the below examples should not be seen aslimiting for the present invention. It is intended that any probioticbacteria may be used in the composition.

In one embodiment of the present invention when the microbe-inhibitingcomposition comprises an extracellular product and at least one of saidadditives, said composition is substantially free from probioticbacteria. It is noted that it is extremely difficult to manufacture acomposition that is totally free from probiotic bacteria, therefore theprobiotic bacteria is preferably not present in the microbe-inhibitingcomposition in an amount higher than 100 CFU/ml and more preferably nothigher than 10 CFU/ml. This composition has the advantage of lessstringent storage requirements since the extracellular product is moredurable than the live micro-organism.

The extracellular product of the present invention is obtained from abacterial culture. Bacteria suitable for the present invention secreteproducts having antimicrobial activity which is useful for theobjectives of the present invention. In order to obtain theextracellular products, cell cultures are harvested as described belowand the supernatant is obtained by centrifugation or filtration or both.The thus obtained supernatant has a microbe inhibiting activity due tothe extracellular products comprised therein. An example of how theextracellular product may be prepared is described under Example 3.

The extracellular product may be in the form as when obtained directlyfrom a bacterial culture but it is also possible to concentrate and/orpurify the supernatant further. By purification is meant that some ofthe microbe-inhibiting substances are isolated by for examplechromatography, crystallisation or distillation and used separately orin combination with other isolated substances.

The additive which is added to the probiotic bacteria and/or to theextracellular product in the present invention is in the form of anorganic acid or a salt thereof. When choosing said additive this shouldof course be made from a product safety point of view. The organic acidsor the corresponding salts should be of such nature that when applyingthem onto a sanitary article they should not cause any skin irritationto the wearer of the sanitary article, therefore the pKa of the organicacid, when the measurement are performed in water at 25° C., shouldpreferably not be lower than 2. If the pKa values instead are too high,the organic ac ids or the salts thereof will mainly be in their acidforms. This means that the additive will not be sufficiently dissociatedinto a hydrogen ion and its deprotonated, anionic form, which isbelieved to be a prerequisite in order to obtain said synergisticmicrobe-inhibiting effect with the probiotic bacteria and/or theextracellular product thereof. Therefore the pKa value for the organicacid suitable for the present invention should be lower than 5.5,preferably lower than 5. If the organic acid suitable for the presentinvention have more than one pKa value, at least one of these valuesshould be lower than 5.5, preferably lower than 5.

When the extracellular product is in the form of a supernatant, thesolubility of these organic acids and/or the corresponding salts shouldbe sufficiently high so that said additive can be dissolved in thesupernatant. Alternatively, when the additive is in a dry/dried form onthe sanitary article the solubility of the additive should be of suchnature that the additive rapidly comes into solution when contacted bythe bodily fluids during use of the sanitary article.

In a preferred embodiment of the present invention the additive ischosen from acetic acid, propionic acid, lactic acid, ascorbic acid,phenylalanine, citric acid, butyric acid, valeric acid, capronic acid,succinic acid and/or a salt thereof. Preferably said additive is chosenfrom acetic acid, propionic acid, lactic acid, ascorbic acid,phenylalanine, citric acid or succinic acid, and/or a salt thereof. Mostpreferably said additive is chosen from ascorbic acid, acetic acid,propionic acid, succinic acid and/or a salt thereof.

In one embodiment of the invention the salt is a sodium salt. Especiallypreferred salts are sodium propionate and sodium acetate.

When a sanitary article of the present invention comprises probioticbacteria these are typically provided in amounts of about 10⁶-10¹¹ CFUof viable probiotic bacteria per article. Typically the bacteria are inthe form of cells or spores which are provided in a suspension which isapplied to the sanitary article which is thereafter dried. Preferably,the sanitary article will comprise about 10⁸-10¹⁰ CFU per sanitaryarticle, although these amounts may vary depending on the specificapplication, product formulation and intended use.

If the sanitary article instead of probiotic bacteria comprises theextracellular product, the sanitary article typically comprises about0.5-100 ml and preferably comprises 1-10 ml of the extracellularproduct.

In combination with the extracellular product said additive is typicallyadded with a concentration of 5-100 mM. When combined only with theprobiotic bacteria said additive is typically added in amounts of 0.15mg-2.6 g and preferably 0.3 mg-0.3 g to the sanitary article, althoughthese amounts may vary depending on the specific application, productformulation and intended use.

The microbe-inhibiting composition may be directly applied to thesanitary article according to the invention or provided in apharmaceutically acceptable carrier which enhances the transfer of themicrobe-inhibiting composition, examples of such includes sticky agentssuch as petrolatum, an oil or a wax.

In one preferred embodiment the microbe inhibiting composition is in theform of a suspension, said suspension may be applied directly to thesanitary article by impregnation, spraying, printing or the like,whereafter the sanitary product is dried, by for e.g. heat drying.

In another preferred embodiment the microbe-inhibiting composition is inthe form of a powder. The powder may be mixed into a pharmaceuticallyacceptable carrier as described above or be directly applied to thesanitary article as a powder, in this latter case preferably to theabsorbent structure or between the absorbent structure and the topsheet, as illustrated in FIG. 5. In the case where the powder is mixedwith a pharmaceutically acceptable carrier, this mixture may be appliedto the sanitary article by impregnation, spraying, printing or the like,whereafter the sanitary article may be dried. A delivery device, meaninga separate pocket, possibly moisture impervious, attached to the topsheet of the sanitary article is also an advantageous way of deliversaid microbe-inhibiting composition.

The powder according to the above may be obtained by evaporating theabove discussed suspension such as by heat drying, convective drying,spray drying, freeze drying, or the like. It may be necessary to furthermould the dried microbe-inhibiting composition in order to obtain a finepowder. The probiotic bacteria are preferably freeze dried.

The microbe inhibiting composition may be placed anywhere on thesanitary article as long as it comes in contact with the user of thesanitary article when in use. The microbe inhibiting composition 8 ispreferably located on the top sheet of the sanitary article as shown inFIG. 4.

In an alternative preferred embodiment, shown in FIG. 5, the microbeinhibiting composition 8 is located between the top sheet 2 and theabsorbent structure 4.

When the sanitary article comprises live probiotic bacteria the bacteriamust be protected from moisture. The sanitary article may therefore behermetically enclosed in a moisture-impervious package. WO 00/76878gives an example of such a moisture impervious package. Another exampleof how the bacteria can be protected is by the use of a delivery systemas exemplified in for example WO 02/28446.

A further possibility is to deliver said microbe-inhibiting compositionseparately in a form which is convenient to apply directly to anabsorbent article by the user, for example in its liquid form, as apowder, dispersed in a lotion, dispersed in oil. The means for applyingsaid microbe-inhibiting composition could be a spray, a roll-on device,a tube, a bottle, an am pull, a strip or a stick.

In a still further aspect of the invention the microbe-inhibitingcomposition is used in combination with the sanitary article accordingto the invention.

Another aspect of the invention is to preparing a sanitary articlecomprising applying said microbe-inhibiting composition to said sanitaryarticle.

Experimental Section

Test 1

The purpose of this test was to determine whether selected additivesaffect the growth of Candida albicans and if an improved growthinhibition can be achieved when they are added to the extracellularproducts of Lactobacillus plantarum, LB 931. The additives used in thistest were ascorbic acid, citric acid, phenylalanine, acetic acid,propionic acid and sodium propionate.

Method

Yeast Strain

A clinical isolate of Candida albicans (designated C. albicans 702) wasused as test strain. It was isolated from the vagina of a woman withvaginal candidiasis.

Control

As control pure dMRSs broth (MRS broth without addition of sodiumacetate) was used. A specified volume was transferred to wells of amicro titre plate and was let to air dry (45° C. minimum of 20 hrs).

Extracellular Product (ECP)

Cultures of LB 931 grown in dMRSs broth were centrifuged to pellet thecells and sterile filtered using a 0.22 μm filter (Millipore filter,Bedford, USA) in order to obtain the LB 931 supernatant comprising theLB 931 extracellular product. A specified volume of sterile filteredsupernatant was transferred to wells of a micro titre plate and was letto air dry (45° C. minimum of 20 hrs) and thereafter resuspended insterile distilled water to a concentration three times higher and 200 μlwere transferred to 96-wells micro titre plates.

Extracellular Product with Additive

The additives were added to a final concentration of 50 mM to tubescontaining the sterile filtered supernatant of LB 931. A specifiedvolume was transferred to wells of a micro titre plate and was let toair dry (45° C. minimum of 20 hrs) and thereafter resuspended in steriledistilled water to a concentration three times higher (i.e. the volumeof the sterile distilled water is about one third of the specifiedvolume before air drying) and 200 μl were transferred to 96-wells microtitre plates.

Broth with Additive

The additives were added to a final concentration of 50 mM in dMRSsbroth. pH was adjusted to the same pH-value as in the extracellularproduct with additive for the respective additive. A specified volumewas transferred to wells of a micro titre plate and was let to air dry(45° C. minimum of 20 hrs) and thereafter resuspended in steriledistilled water to a concentration three times higher and 200 μl wastransferred to 96-wells micro titre plates.

Candida was added to a final concentration of ˜10⁴ cells per well andthe micro titre plate was incubated at 37° C. for 24 hrs.

pH for the different additives varied between 3.4±0.2. The pH for brothwith additive and extracellular product with additive was always thesame.

The test was blinded so that there could be no preconceived opinionscolouring the results. The inhibition of Candida was evaluated andgraduated, by two persons using a template, on a scale from five to zerobased on visual observations of turbidity with a calculated mean valuebeing presented. Wells containing strong growth of Candida in puredMRSs-broth was graduated as five, while wells with no visual growthwere graduated as zero.

Results

As can be seen in FIG. 1 very few additives themselves proved to have aneffect on the growth of Candida although the pH was very low. Theextracellular product of LB 931 was effective in the inhibition ofCandida. This effect was surprisingly enhanced by the addition of theadditives to the extracellular products of LB 931. For all additives incombination with the extracellular product of LB931, a very good growthinhibiting effect was achieved. The best effect was achieved whenpropionic acid was added to the extracellular product of LB931 (nogrowth at all).

Test 2

The purpose of these tests was to determine whether selected additivesaffect the growth of bacteria and if an improved growth inhibition canbe achieved when they are added to the extracellular products ofLactobacillus plantarum, LB 931. The additives used in this test weresodium acetate, succinic acid, sodium propionate, acetic acid.

Method

Bacterial Strains

Clinical isolates of E. coli (designated E. coli 1) and Staphylococcussaprophyticus (S. saprophyticus 1) were used as test strains. They wereisolated from the genital tract in women with urinary tract infections.

Control

As a control pure dM17s broth (M17 broth modified by addition of MnSO₄(final conc. 0.04 g/l), MgSO₄ (final conc. 0.2 g/l) and glucose (finalconc. 20 g/l) was used. 200 μl was added to 96-wells micro titre plates.

Extracellular Product (ECP)

Cultures of LB 931 grown in dM17s broth were centrifuged to pellet thecells and sterile filtered using a 0.22 μm filter (Millipore filter,Bedford, USA) in order to obtain the LB 931 supernatant comprising theLB 931 extracellular product. 200 μl were transferred to 96-wells microtitre plates.

Extracellular Product with Additive

The additives were added to a final concentration of 50 mM to tubescontaining the sterile filtered suspension of LB 931. 200 μl wastransferred to 96-wells micro titre plates.

Broth with Additive

The additives were added to a final concentration of 50 mM in dM17sbroth. 200 μl were added to 96-wells micro titre plates.

10 μl of an over-night culture of E. coli resp. S. saprophyticus, bothdiluted 100×, were added to the wells in the micro titre plate which wasincubated in 37° C. over night.

pH was adjusted to 6.9 in all trials.

The test was blinded so that there could be no preconceived opinionscolouring the results. The inhibition of E. coli resp. S. saprophyticuswas evaluated and graduated, by two persons using a template, on a scalefrom five to zero based on visual observations of turbidity with acalculated mean value being presented. Wells containing strong growth ofE. coli resp. S. saprophyticus in pure dM17s-broth was graduated asfive, while wells with no visual growth were graduated as zero.

Results

As can be seen in FIG. 2A none of the tested additives alone proved tohave an effect on the growth of E. coli. The extracellular product of LB931 was effective in the inhibition of E. coli. This effect wassurprisingly enhanced by the addition of the additives to theextracellular products of LB 931. For all additives in combination withthe extracellular product of LB931, a very good growth inhibiting effectwas achieved.

As can be seen in FIG. 2B none of the tested additives alone proved tohave an effect on the growth of Staphylococcus saprophyticus. Theextracellular product of LB 931 was effective in the inhibition ofStaphylococcus saprophyticus. This effect was surprisingly enhanced bythe addition of the additives to the extracellular products of LB 931.For all additives in combination with the extracellular product of LB931, a very good growth inhibiting effect was achieved.

Test 3

The purpose of this test was to evaluate the enhanced inhibition ofCandida albicans when extracellular product of the Lactobacillusfermentum Ess-1 was combined with the additives ascorbic acid orpropionic acid.

Method

Ess-1 was grown to stationary phase in dMRSs broth (MRS broth withoutaddition of sodium acetate). The bacterial culture was centrifuged topellet the cells and sterile filtered using a 0.22 μm filter (Milliporefilter, Bedford, USA) whereupon the supernatant comprising theextracellular product was obtained. Propionic acid and ascorbic acidwere added to a final concentration of 50 mM to tubes containing theextracellular product. The filtrate was pH adjusted to the pKa-value forrespective acid (4.2 for ascorbic acid and 4.87 for propionic acid) and200 μl were transferred to wells of a 96-wells micro titre plate. C.albicans was added to a final concentration of ˜5×10⁴ CFU ml⁻¹ to eachwells. The plates were incubated for 20 hours at 37° C. and the growthwas evaluated and graduated, by two persons using a template, on a scalefrom five to zero based on visual observations of turbidity with acalculated mean value being presented. Wells containing strong growth ofCandida in pure dMRSs-broth was graduated as five, while wells with novisual growth were graduated as zero.

Results

An unexpected increased inhibition was obtained when the supernatantfrom Ess-1 was combined with either ascorbic acid or propionic acid ascan be seen in FIGS. 3A and 3B respectively.

Test 4

The purpose of this test was to evaluate the enhanced Candida inhibitionfrom Na-acetate in combination with growing cells of Lactobacillusplantarum LB 931.

Method

Two plates were made with M17 agar and two with MRS agar. MRS medium(developed by De Man, Rogosa and Sharpe), is a growth medium commonlyused for Lactobacillus. MRS agar from Merck contains 5 g sodium acetateper litre agar. M17 (Oxoid) is a similar commercial substrate forLactobacillus but without the addition of sodium acetate. The agars wereprepared as described on the packages.

Lactobacillus plantarum LB931 was cultivated in MRS broth in 37° C. with5% CO₂ for 24 hours. Candida albicans 702 was cultivated in M17 broth at37° C. for 24 hours.

Holes (Ø10 mm) were punched in the plates and filled with 250 μl plugsof agar mixtures containing ˜10⁷ LB 931 in MRS agar). The plates wereleft for 30 minutes and then incubated 24 hours at 37° C. with 5% CO₂.Overnight culture of C. albicans in M17 broth was diluted 10 times and100 μl was spread on the surface of each plate. The plates were thenincubated for 24 hours at 37° C. As controls, holes (Ø10 mm) were filledwith only MRS agar.

The plates were evaluated in respect to Candida growth inhibition. Zonesaround the plugs with no growth of Candida were measured (Ø mm).

Results

The sizes of the zones can be seen in Table 1

TABLE 1 Agar in the Plug with Plug with Petri dish only agar (Ø mm)LB931 (Ø mm) MRS agar 0 18.8 M17 agar 0 0 MRS agar 0 18.1 M17 agar 0 0

LB 931 grew well in all plugs where LB 931 had been inoculated. Therewere no zones without growth of Candida on the plates with M17 agar. Onthe MRS agar plates there were clear zones around the LB 931 plugs butno zones around the plugs with only agar.

LB 931 was applied in quite low amounts to the plates, which may explainwhy substantially no inhibition of Candida was seen on the M17 agarplates. This however indicates the efficient inhibition of Candida dueto the synergistic effect of LB 931 and the additive, in this casesodium acetate comprised in the MRS medium.

EXAMPLES

The following examples related to this invention are illustrative andshould not, of course, be construed as specifically limiting theinvention.

Example 1 Isolation and Typing of Ess-1

The initial purpose of this study is to isolate and type a Lactobacillusstrain that inhibit the growth of Candida albicans and Candida glabratato a large extent compared to other Lactobacillus strains.

Method

Yeast Strains

Clinical isolates of Candida albicans and Candida glabrata were used astest strains. These were isolated from the vagina of women with vaginalcandidiasis and from healthy females.

Screening I.

About 140 Lactobacillus strains, originating from human skin, throat,teeth, baby faeces, vegetables and seeds were cultured in MRS broth andstamped onto MRS agar plates. The agar plates were incubated underanaerobic conditions at 37° C. Additionally, SAB (Sabouraud) agar (LABM, Bury, UK) was poured onto the MRS agar and was allowed to congeal. C.albicans culture was seeded onto the agar and the plates were incubatedaerobically at 37° C. A visual evaluation of the inhibition was done.Strains inhibiting C. albicans equally or to a greater extent than thereference strain Lactobacillus plantarum LB931 were selected for furtherscreening (screening II).

Screening II.

Suspensions of lactobacilli grown in dMRSs broth (MRS broth withoutaddition of sodium acetate) were centrifuged and sterile filtered. Thefiltrate is henceforth called Lactobacillus Cell-free Filtrate, LCF. Aspecified volume was transferred to wells of a micro titre plate and waslet to air dry and thereafter resuspended in sterile distilled water toa concentration three times higher and transferred to 96-wells microtitre plates. Candida was added to all vials (three isolates of C.albicans and C. glabrata, respectively, were used). The inhibition wasevaluated by visual observation of turbidity and graduated by twopersons using a template on a scale from five to zero. The wellscontaining strong growth of Candida sp. in pure dMRSs-broth wasgraduated as five, while wells with no visual growth were graduated aszero.

API Typing and Genetic Typing

Identification to the species level was done using the API 50 CHL system(bioMérieux, France), following the manufacturer's instructions. Datafrom the fermentation tests were analysed using API Lab Plus software.Genetic typing was done by DSMZ (Deutsche Sammlung von Microorganismenund Zellkulturen GmbH) by partial sequence analysis of the 16S rRNA.

Results

All strains were evaluated according to the growth inhibition capacityagainst C. albicans in screening I. 23 Lactobacillus strains withresults comparable or exceeding the one for LB931 were selected forscreening II. Out of those 23 Lactobacillus strains, three werereference strains and a majority of the strains had been isolated fromthe oral tract.

Ess-1 proved to have a comprehensive capacity to inhibit growth ofCandida. No one of the tested Lactobacillus strains was similar to theactivity of Ess-1 regarding the effect on both C. albicans and C.glabrata. The carbohydrate fermentation pattern and the genetic typingfor Ess-1 showed that it belongs to the Lactobacillus fermentum species.

Characterization of Ess-1

16S rDNA from strain Ess-1 (DSM 17851) was analysed by DSMZ (DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH) by direct sequencingof about 450 nucleotides of PCR-amplified 16S rDNA. Genomic DNAextraction, PCR mediated amplification of the 16S rDNA and purificationof the PCR product was carried our as previously described (Rainey, F.A. et al. Int. J. Sys. Bacteriol. 1996(46):1088-1092). Purified PCRproducts were sequenced using the CEQ™DTCS-Quickstart Kit (BeckamnCoulter) following the manufacturers instructions. Sequence reactionproducts were electrophoresed using the CEQ™8000 Genetic AnalysisSystem.

The resulting sequence data was put into the alignment editor ae2(Maidak, B. L. et al. Nucl. Acids Res. 1999(27):171-173), alignedmanually and compared with representative 16S rRNA gene sequences oforganism belonging to the Firmicutes (Maidak, B. L. et al.). Forcomparison 16S rRNA sequences were obtained from the EMBL data base orRDP (Maidak, B. L. et al.).

As a result of this analysis the following table 2 lists thoseorganisms, whose 16S rRNA gene sequences show the highest similarityvalues compared to the 16S rDNA sequence of Ess-1.

TABLE 2 % 16S rRNA gene sequence Strain similarity to Ess-1Lactobacillus fermentum ATCC 14931 99.3 Lactobacillus fermentum KRM 99.0Lactobacillus malefermentans DSM 5705 85.5 Lactobacillus rossiae ATCCBAA-822 88.9 Lactobacillus suebicus DSM 5007 86.9 Lactobacillusvaccinostercus DSM 20634 87.2

The partial 16S rDNA gene sequence of strain Ess-1 shows highestsimilarity to Lactobacillus fermentum. Consequently strain Ess-1 mayrepresent a strain of Lactobacillus fermentum, but may as well representa new species within the genus Lactobacillus.

Example 2 Production of Probiotic Lactobacillus

In laboratory scale: a sterile vessel with ex. MRS broth is inoculatedwith a pure inoculate of the desired lactobacillus strain. Broth withbacteria is incubated over night in 37 C.° and preferably with anatmosphere of 5% CO₂. Cells may then be washed using centrifugation andsterile saline. Protective sugar, starch or proteins may be added andthe cell-mass freeze-dried and milled.

In production scale, the working sead lot is fermented in differentsteps up to production scale. The production is controlled and optimisedfor the specific strain to be produced. After fermentation the cultureis washed and/or only concentrated using cross-flow microfiltration orcentrifugation. Protective substances according to the above are addedand the cells can be freeze-dried and milled or spray-dried.

Example 3 Production of Extracellular Product from e.g. Lactobacillus

In laboratory scale the overnight culture of the probiotic strain isproduced as in Example 2. After filtration or centrifugation thesupernatant is collected. The supernatant may be concentrated byevaporation or completely dried for ex. convective drying in dry air,freeze, drying or spray-drying.

In production scale the bacterial culture is fermented in accordancewith Example 2. The extracellular product is obtained as a filtrateusing cross-flow micro filtration or a supernatant using centrifugation.Also in this case it may be further concentrated using evaporation orcompletely dry using for eg. spray-drying.

Example 4 Formulations

Anti Candida Panty Liner

The product is based on a standard marketed panty liner, such asLibresse® extra liners (sold by SCA Hygiene Products AB), to which theextracellular product of Ess-1 in combination with an additive, is addedaccording to the following: 10 ml of the extracellular product of Ess-1is evaporated to 1 ml to which 3.7 mg of propionic acid is added. Theextracellular product with propionic acid is printed on the surface ofthe panty liner which is dried before packaging.

Light Incontinence Pad with Reduced Bacterial Growth and Odour Control

The product is based on a standard marketed light incontinence pad, suchas Tena® Lady Normal (sold by SCA Hygiene Products AB), to which theextracellular products of LB931 is added according to the following: 100ml of the extracellular product of LB931 are mixed with 1 g of citricacid. The mixture is evaporated and dried and thereafter the powder ishomogenously mixed in the absorbent core of the pad.

Light Liner for Improved Genital Hygiene

The product is based on a standard marketed panty liner, such asLibresse® light liners (sold by SCA Hygiene products AB), to which theextracellular products of both Ess-1 and LB931 are added according tothe following: 5 ml of the extracellular product of Ess-1 and 5 ml ofthe extracellular product of LB931 are mixed and evaporated to 1 ml, towhich 4.1 mg of sodium acetate are added. The mixture is evaporated anddried and the powder is mixed with 0.1 g of silicone wax and finallyprinted in the centre of the top sheet of the liner.

It should be noted that even if all three examples on formulations onlycomprise Lactobacillus plantarum LB 931 and/or Lactobacillus fermentumEss-1 as probiotic bacteria, all probiotic bacteria suitable for thepresent invention as previously described may as well be used.

The invention claimed is:
 1. A sanitary article comprising a top sheet,said sanitary article comprising, prior to use of the sanitary article:a microbe-inhibiting composition, said microbe-inhibiting compositioncomprising: an extracellular product of at least one probioticbacterium, or at least one probiotic bacterium, and at least oneadditive in the form of an organic acid, having a pKa value notexceeding 5.5, or a salt of the organic acid, wherein, when themicrobe-inhibiting composition comprises the extracellular product, theadditive is present in a concentration of 5-100 mM, and when themicrobe-inhibiting composition comprises the probiotic bacteriaexcluding the extracellular product thereof, the additive is present inan amount of 0.15 mg-2.6 g per the sanitary article.
 2. The sanitaryarticle according to claim 1, wherein said pKa value does not exceed 5.3. The sanitary article according to claim 1, wherein said extracellularproduct is in the form of a supernatant, obtained by centrifugation orfiltration of a culture of a probiotic bacterium.
 4. The sanitaryarticle according to claim 1, wherein said microbe-inhibitingcomposition comprises at least one probiotic bacterium and at least oneof said additives.
 5. The sanitary article according to claim 1, whereinsaid microbe-inhibiting composition comprises at least one probioticbacterium, an extracellular product of at least one probiotic bacteriumand at least one of said additives.
 6. The sanitary article according toclaim 1, wherein said microbe-inhibiting composition is substantiallyfree from probiotic bacteria.
 7. The sanitary article according to claim1, wherein said probiotic bacterium is a lactic acid producingbacterium.
 8. The sanitary article according to claim 7, wherein saidlactic acid producing bacterium is Lactobacillus plantarum LB 931 orLactobacillus fermentum Ess-1 or a combination thereof.
 9. The sanitaryarticle according to claim 1, wherein said additive is selected from thegroup consisting of acetic acid, propionic acid, lactic acid, ascorbicacid, phenylalanine, citric acid, butyric acid, valeric acid, capronicacid, succinic acid and salts thereof.
 10. The sanitary articleaccording to claim 9, wherein said additive is a sodium salt of aceticacid, propionic acid, lactic acid, ascorbic acid, phenylalanine, citricacid, butyric acid, valeric acid, capronic acid, or succinic acid. 11.The sanitary article comprising a microbe-inhibiting compositionaccording to claim 1, wherein the microbe-inhibiting composition islocated on the top sheet.
 12. A kit comprising, prior to use of the kit:a microbe-inhibiting composition comprising: an extracellular product ofat least one probiotic bacterium, or at least one probiotic bacterium,and at least one additive in the form of an organic acid, having a pKavalue not exceeding 5.5, or a salt of the organic acid, and a sanitaryarticle, wherein when the microbe-inhibiting composition comprises theextracellular product, the additive is present in a concentration of5-100 mM, and when the microbe-inhibiting composition comprises theprobiotic bacteria excluding the extracellular product thereof, theadditive is present in an amount of 0.15 mg-2.6 g per the sanitaryarticle.
 13. The kit according to claim 12, wherein saidmicrobe-inhibiting composition is provided in an ampoule, a bottle, atube, a roll-on device, as a stick or a spray.
 14. A method of preparinga sanitary article according to claim 1 comprising applying saidmicrobe-inhibiting composition to said sanitary article.
 15. Thesanitary article according to claim 6, wherein the probiotic bacteriaare present in an amount not higher than 10 CFU/ml.
 16. The sanitaryarticle according to claim 9, wherein said additive is selected from thegroup consisting of ascorbic acid, acetic acid, propionic acid, succinicacid and salts thereof.
 17. The sanitary article according to claim 10,wherein said salt is sodium propionate or sodium acetate.